LAP-MALDI MS Profiling and Identification of Potential Biomarkers for the Detection of Bovine Tuberculosis

Detecting bovine tuberculosis (bTB) primarily relies on the tuberculin skin test, requiring two separate animal handling events with a period of incubation time (normally 3 days) between them. Here, we present the use of liquid atmospheric pressure (LAP)-MALDI for the identification of bTB infection, employing a three-class prediction model that was obtained by supervised linear discriminant analysis (LDA) and tested with bovine mastitis samples as disease-positive controls. Noninvasive collection of nasal swabs was used to collect samples, which were subsequently subjected to a short (<4 h) sample preparation method. Cross-validation of the three-class LDA model from the processed nasal swabs provided a sensitivity of 75.0% and specificity of 90.1%, with an overall classification accuracy of 85.7%. These values are comparable to those for the skin test, showing that LAP-MALDI MS has the potential to provide an alternative single-visit diagnostic platform that can detect bTB within the same day of sampling.


Contents
Table S1 -  The purpose of this document is to provide guidance for sample collection of oral and nasal swabs from cattle for the further downstream processing and analysis by mass spectrometry.

Scope
This SOP is designed for the appropriate collection of nasal and oral swabs from cattle for disease diagnostics.

Health and Safety
Handle all swab samples as a potential source of pathogens, use appropriate personal protective equipment and handle samples as potential carriers of (bio)hazardous materials.Users of this protocol should have read and understood all relevant Risk and COSHH Assessments and other relevant SOPs, in particular health and safety rules and regulations and SOPs for handling farm animals and their body fluids.Before attempting to use this SOP, it is important to read and understand this document in its entirety.

Personnel required
-Two trained research, farm or veterinary staff will be required for the whole duration of each sampling.This is necessary to ensure the safety of personnel and the safety and welfare of the animals being sampled.

Procedural guidelines
Only collect oral and nasal swabs whilst the animal is physically restrained in a cattle crush.
The animal should then be haltered and tied to the side of the crush in such a way that the head cannot move.This is essential both for the safety of the staff taking the swabs and for the safety of the animal as there is a danger the swab could break off in nasal cavity or mouth/pharynx.Only experienced staff members should attempt to restrain a cow as inexperience leads to dangers for all involved.
Once the animal is secure within the crush and correctly haltered, the procedure of taking the swab can be followed.

5.1.1
Clean nostrils provide the best nasal samples.However, traces of dirt or feed in the animal's nostrils is acceptable though not desirable.Wherever possible, avoid collecting swab samples immediately after the animal has eaten.Ensure the collection end of the swab does not touch anything other than the animal's nostril to prevent contamination of the sample.

5.1.2
Holding the tube, unscrew the lid and carefully remove the swab.Immediately, rub the end containing the swab inside the animal's nostril for 3-5 seconds.
Ensure the swab looks wet and is coated with the nasal sample to achieve best results.

5.1.3
Holding the tube in one hand, carefully insert the nasal swab into the tube.Screw cap on tightly to prevent any leakage during transport.

5.1.4
Using a permanent marker, clearly write the animal identification number, farm identification code and the type of swab (nasal or oral) on the side of the tube and place the tube in a grip seal bag and immediately on ice.

5.1.5
Go to 5.2 for oral swab sample collection.

5.2.1
As before, avoid collecting swab samples immediately after the animal has eaten.
Try to avoid traces of dirt or feed.Ensure the collection end of the swab does not touch anything other than the animal's mouth to prevent contamination of the sample.

5.2.2
Holding the tube, unscrew the lid and carefully remove the swab.Immediately, rub the end containing the swab on the inside base of the animal's mouth to coat in saliva for 5 seconds.Please note that too much pressure can result in swab breaking.Ensure the swab looks wet and is coated with the saliva sample to achieve best results.

5.2.3
Holding the tube in one hand, carefully insert the swab into the tube.Screw cap on tightly to prevent any leakage during transport.

5.2.4
Using a permanent marker, clearly write the animal identification number, farm identification code and the type of swab (nasal or oral) on the side of the tube and place the tube in a grip seal bag and immediately on ice.

5.3.1
Following swab sample collection, the grip seal bags containing the swabs are placed into an ice-filled freezer transport box with the lid securely on for transportation.

5.4.1
Trained and vaccinated personnel will receive samples delivered to the Chemistry building and transport them straight to laboratory room 142 in the Chemistry building.

5.4.2
For samples to be stored, swabs will be stored in sample quarantine within a storage freezer box in a locked -80°C freezer in laboratory room 142 until required for sample processing.

5.4.3
For further processing, samples are moved into a microbiological safety cabinet where the packaging can be removed, making the sample accessible for downstream processing.Follow all relevant risk assessment for handling and further processing of the samples.The purpose of this document is to provide guidance for the processing of nasal swabs from cattle for analysis by liquid atmospheric pressure (LAP)-MALDI mass spectrometry (MS), and the subsequent data analysis and mining, for the detection of bovine tuberculosis (bTB).

Scope
This SOP has been designed for the appropriate processing of nasal swabs from cattle for the detection of bTB and potentially other diseases.

Health and Safety
Swab samples to be used following this SOP will contain pathogens including causative agents of bTB (Mycobacterium bovis) and bovine mastitis (including but not limited to Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli and Streptococcus uberis).M. bovis is a biosafety level 3 bacterium and therefore must be handled in a category 3 laboratory.A derogation from category 3 to category 2 for the handling of M. bovis containing samples can be applied for from HSE providing suitable health and safety measures.Bacterial species causing bovine mastitis are biosafety level 2 pathogens and can therefore be handled in a category 2 facility.All swab samples, whether health status is known or not, should be handled as a potential source of pathogens, using appropriate personal protective equipment, and handled as potential carriers of (bio)hazardous materials.Users of this protocol should have read and understood all relevant Risk and COSHH Assessments and other relevant SOPs, in particular health and safety rules and regulations and SOPs for handling farm animals and their body fluids.Before attempting to use this SOP, it is important to read and understand this document in its entirety and ensure all suitable H&S procedures are in place before commencing.

Procedural guidelines
These procedures are for the preparation of bovine nasal swab samples collected as detailed in the SOP for oral and nasal swab collection for the diagnosis of bovine tuberculosis (bTB), v1.3.Swabs can be either directly analysed using this method, or thawed from quarantine in the -80°C freezer.
Note: It is advisable that all steps will be carried out by keeping the samples and extracts etc. on ice as much as possible.If storage time is needed between the various sample preparation stages, this should be minimized and kept the same within a (disease classification) study.For longer storage times, the samples should be kept in a -80°C freezer.

a. Inactivation and Precipitation
i. Move the swabs to the microbiological safety cabinet (MSC), either from sample quarantine or directly from the transportation packaging.ii.Inside the MSC, carefully remove swab from its casing and place it into a 1.5-mL tube containing 400µL of 1X PBS, and briefly agitate (at least five times).iii.Discard swab after squeezing it gently against the inside walls of the 1.5-mL tube.iv.Add 900µL of 100% ethanol and vortex to mix.Samples should be left on ice if multiple samples are processed at the same time.v. Centrifuge samples for 5 minutes at 13,000 rpm.Remove and discard the supernatant and resuspend the resultant pellet in 30µL of 0.1% TFA.

b. Trypsin Digestion
i. Add 50µL of 50mM ammonium bicarbonate to the samples and mix by pipetting.ii.Add 5µL of 100mM DTT and vortex.Incubate at 37°C for 30 minutes.iii.Add 10µL of 100mM IAA and vortex.Incubate at room temperature in the dark for 30 minutes.iv.Add 2µL of sequencing grade trypsin (0.2µg/µL) and incubate at 37°C for 2 hours.v. Add 8µL of 10% TFA to inactivate trypsin and stop the reaction.vi.Purify the samples and extract the peptides by using ZipTips according to the manufacturer's instructions, with a final elution volume of 5µL.

c. Liquid MALDI MS Sample Preparation
i. Prepare a liquid support matrix (LSM) for MALDI analysis by dissolving CHCA in 70:30 ACN:H 2 O at a concentration of 25mg/mL.The solution can be sonicated in an ultrasonic water bath to assist dissolving the CHCA crystals.Once fully dissolved, add PG (by adding 7 volume parts of PG to 10 volume parts of CHCA solution) and mix by vortexing.ii.For each sample, spot 0.5µL of the LSM on to a stainless steel MALDI sample plate followed by the addition of 0.5µL of the digest sample to the same spot.iii.Mount the MALDI sample plate on to the plate holder.

d. MS Data Acquisition
Note: The following steps and instrument settings are just for general guidance for liquid AP-MALDI MS analysis of samples as prepared in the previous steps on a Waters Synapt G2-Si instrument.
i. Use the following instrument modes: Mobility TOF, Positive Ion, Sensitivity and MS.
ii. Calibration of the instrument should be performed in Mobility TOF MS mode using sodium iodide over the m/z range of 100-2000.
iii.Set the laser energy to 18µJ/pulse and the laser pulse repetition frequency to 30Hz, continuous pulsing.

Note:
The exact threshold laser energy that is best for an effective detection of analyte ion signals depends on the laser focus on the droplet surface and other ion source parameters (e.g.laser beam angle/position on droplet).Details about automatic data acquisition and the associated sample stage movement depend on the exact plate size and analytes to be detected, amongst other ion source parameters and analytical objectives.Thus, no further details on automatic data acquisition will be given.

e. Data Analysis
For sample classification, there are many types of software that can be used for statistical modelling.In this case, we have used Abstract Model Builder (AMX, Waters .This can also take up to 30 minutes.xiv.Click 'OK' when the 'Cross validation terminated successfully.The report can be seen on the reports page.' window appears.xv.Within the 'Cross validation' tab, switch to the 'Reports' tab.Select the report required, which can be identified by the name and creation time.Selecting the report will display the confusion matrix, the correct classification rate and the identification of outliers.xvi.The cross validation report can be saved by clicking 'Save…' on the appropriate report.By default, this is saved as a .csvfile.Click on 'Save' and subsequently on 'OK' when the 'CSV file generated successfully.'window appears.xvii.To export the data matrix for use in other applications, switch to the 'Home' tab and select 'Export data matrix' in the top toolbar.This will export the data matrix according to the settings applied in the 'Model properties' tab as described in 5.5.6-5.5.10.This is exported as a .csvfile.xviii.When leaving the software, don't forget to save the current project, following the prompts in the various windows that will appear., P., et al. (2016)."Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS." Methods 104: 11-20.

Ryumin
Figure S1 -LAP-MALDI Mass Spectrum of a bTB Sample

SOP for Nasal Swab Sample Collection Standard Operating Procedure (SOP) for oral and nasal swab collection for the diagnosis of bovine tuberculosis (bTB) Version 1.3 (24 th November 2021) Barney Jones, Sophie Lellman and Rainer Cramer Department of Chemistry, UoR 1. Purpose
Sample collection is performed by trained research, farm or veterinary staff, in many cases at the time of reading the tuberculin skin test.In order to efficiently detect biomarkers for indicators of disease it is essential that a robust sample preparation protocol is established.In some cases, these biomarkers may be present in very small quantities.The presence of an established SOP will minimise the effects of user variability during sample collection.

4. Recommended equipment/material and personnel required (per collection event)
Nasal swabs have been previously collected by trained research, farm or veterinary staff (see Standard Operating Procedure (SOP) for oral and nasal swab collection for the diagnosis of bovine tuberculosis (bTB) -version 1.3).The sample preparation protocol has been optimised to include a short digestion step to assist in the detection of biomarkers for disease classification.The following protocol is designed for data acquisition using LAP-MALDI MS and subsequent data analysis.
Disclaimer: This protocol has been developed and tested for research use only, not for veterinary or clinical use.No claims are made for its usefulness, accuracy or safety, and no liability can be accepted for any damages, losses or other expenses of any nature whatsoever arising from its use or supply.This protocol does not cover any legal or ethical issues.
).A training set of LAP-MALDI mass spectral profiles was generated for classification of healthy, mastitis and tuberculosis samples.Cross-validation of the model was performed in the absence of unknown samples to test against the model, whereby 20% of the training set data was left out and tested against the remaining data set. .Click on 'Add samples', which takes you back to the 'Sample list' tab.iv.Select all files and in the 'TIC Operations' toolbar, click on 'Spectrum from all scans'.v. Switch to the 'Model properties' tab.vi.Enter an appropriate model name in the 'Model name' field and select 'LDA model' from the 'Model type' drop-down menu.vii.In the 'PCA dimensions' field, set the value to 10 (or less if you have less .rawfiles) and in the 'LDA dimensions' field, set the value to n-1 (n = number of classes).In the 'Intensity limit' field, enter 1.00E2.viii.Binning is kept at default values, performed every 1 Da in the mass range of 650-2000 in advanced mode.ix.Under 'Spectrum interpretation' select 'One spectrum per burn'.x.Under 'Preprocessing' only select 'Apply background subtraction' and 'Apply normalization'.xi.Switch to the 'Model builder' tab and click on 'Build the model'.This can take up to 30 minutes, if a high number of data files are provided.xii.Once the model building is complete, switch to the 'Visualisation' tab for a representation of the PCA/LDA model.By default, the first 3 components that are most responsible for variation are displayed.This can be altered by clicking the '…' icon on the top toolbar, to the right of the text field, which indicates the displayed components.xiii.Switch to the 'Cross validation' tab.Keep the default settings the same, which are the '20% out' function, with the 'Outlier' drop down menu set to 'Based on standard deviation' and the 'Std.dev.multiplier' set to 5. Enter a suitable report name in the 'New report name' field and click on 'Start' ii. Switch to the 'Sample List' tab.Click 'Add sample files' and select all Waters .rawfiles to be added to the model, click on the single arrow tab and apply the correct classification to each file according to the sample type (healthy, mastitis, reactor).iii